Confocal microscope pinhole

In a confocal microscope, invented in the mid-1950s, a focused spot of light scans the specimen. The fluorescence emitted by the specimen is separated from the incident beam by a dichroic mirror and is focused by the objective lens through a pinhole aperture to a photomultiplier. Fluorescence from out-of-focus planes above and below the specimen strikes the wall of the aperture and cannot pass through the pinhole (Figure 11.3). 

Fig. 1. Comparisons of the wide-field, flying spot, pinhole detector, and pinhole confocal microscopes. Components include an excitation light source (V), an excitation filter (E), a dichromatic mirror (DM), an emission barrier filter (B), an objective lens (n), a detector (D), and a pinhole (P).

Fig. 2. Depth discrimination (z-axis resolution) properties of a confocal microscope. The illumination and detection images in a confocal microscope are diffraction-limited and confined to a small region of the specimen (1). Only light emitted in the plane of focus and on the optical axis will pass the detector pinhole and form an image. Light emitted from other areas of the specimen does not enter the detector pinhole.

Use of a confocal microscope (46) greatly improves Raman microspectroscopy. As shown in Fig. 3-12, confocal microscope has a pinhole (25 to 100 urn in diameter) which rejects out-of-focus signals. The smaller the pinhole size, the better the rejection. As a result, the background (substrate) signals can be... 

Laser scanning confocal microscope A fluorescence microscope achieving improved depth discrimination and contrast by blocking fluorescence that originates outside the plane of focus by use of a confocal pinhole. 

In transmission confocal microscopes that are equipped with phasereading systems, the focus deviates from the pinhole because of the inhomogeneity of the refractive index and/or the thickness of the memory medium and substrate. As a result, the detected signal has a background owing to the local inhomogeneity of the medium and substrate. 

Fig. 3. Simplified diagram of the confocal components of the AC AS 570. The laser beam is expanded to provide a diffraction limited spot at the sample. A focusing lens in front of the detection system directs the fluorescence from the focal plane through an adjustable pinhole. In the bilateral laser scanning confocal microscope, a similar technique is employed with a cursor rather than a point for illumination and detection.

Figure 12. Schematic diagram of the scanning laser confocal microscope. The out-of-focus information that normally reaches the eyepiece (detector) and leads to difficulties in interpreting optical images is rejected because the optical path does not take it through the pinhole. By scanning the incident laser beam across the sample, a digitized image is constructed from the infocus light rays that pass through the pinhole.

Fig. 5.72 Intensity distribution around the laser focus, seen from the surface of a thick sample. Left One-photon excitation. Fluorescence comes from the complete excitation light cone. The confocal microscope obtains a sharp image by detecting through a pinhole. Right Two-photon excitation. Fluorescence is excited only in the focal plane. Nevertheless, scattering in a thick sample blurs the image seen from the sample surface. The two photon microscope obtains a sharp image by assigning all photons to the pixel in the current scan position...

Confocal microscopes provide particularly good spatial resolution. They are mainly used in Raman and fluorescence measurements. The basic idea of a confocal microscope is that all structures being out of focus are suppressed at the detector. This is achieved by point illumination and a pinhole in front of the detector. The optical layout of a confocal microscope is shown in Fig. 5.15. 

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